Talvinder S. Sihra

Synapsins as mediators of BDNF-enhanced neurotransmitter release.

We examined enhancement of synaptic transmission by neurotrophins at the presynaptic level. In a synaptosomal preparation, brain-derived neurotrophic factor (BDNF) increased mitogen-activated protein (MAP) kinase-dependent synapsin I phosphorylation and acutely facilitated evoked glutamate release. PD98059, used to inhibit MAP kinase activity, markedly decreased synapsin I phosphorylation and concomitantly reduced neurotransmitter release. The stimulation of glutamate release by BDNF was strongly attenuated in mice lacking synapsin I and/or synapsin II. These results indicate a causal link of synapsin phosphorylation via BDNF, TrkB receptors and MAP kinase with downstream facilitation of neurotransmitter release.

Presynaptic kainate receptor facilitation of glutamate release involves protein kinase A in the rat hippocampus

We have explored the mechanisms involved in the facilitation of glutamate release mediated by the activation of kainate receptors in the rat hippocampus using isolated nerve terminal (synaptosome) and slice preparations. In hippocampal nerve terminals, kainate (KA) produced an increase of glutamate release at concentrations of agonist ranging from 10 to 1000 μm. In hippocampal slices, KA at low nanomolar concentrations (20–50 nm) also produced an increase of evoked excitatory postsynaptic currents (eEPSCs) at mossy fibre–CA3 synapses. In both, synaptosomes and slices, the effect of KA was antagonized by CNQX, and persisted after pretreatment with a cocktail of antagonists for other receptors whose activation could potentially have produced facilitation of release. These data indicate that the facilitation of glutamate release observed is mediated by the activation of presynaptic glutamate receptors of the kainate type. Mechanistically, the observed effects of KA appear to be the same in synaptosomal and slice preparations. Thus, the effect of KA on glutamate release and mossy fibre–CA3 synaptic transmission was occluded by the stimulation of adenylyl cyclase by forskolin and suppressed by the inhibition of protein kinase A by H‐89 or Rp‐Br‐cAMP. We conclude that kainate receptors present at presynaptic terminals in the rat hippocampus mediate the facilitation of glutamate release through a mechanism involving the activation of an adenylyl cyclase–second messenger cAMP–protein kinase A signalling cascade.

Presynaptic signaling by heterotrimeric G-proteins.

G-proteins (guanine nucleotide-binding proteins) are membrane-attached proteins composed of three subunits, alpha, beta, and gamma. They transduce signals from G-protein coupled receptors (GPCRs) to target effector proteins. The agonistactivated receptor induces a conformational change in the G-protein trimer so that the alpha-subunit binds GTP in exchange for GDP and alpha-GTP, and betagamma-subunits separate to interact with the target effector. Effector-interaction is terminated by the alpha-subunit GTPase activity, whereby bound GTP is hydrolyzed to GDP. This is accelerated in situ by RGS proteins, acting as GTPase-activating proteins (GAPs). Galpha-GDP and Gbetagamma then reassociate to form the Galphabetagamma trimer. G-proteins primarily involved in the modulation of neurotransmitter release are G(o), G(q) and G(s). G(o) mediates the widespread presynaptic auto-inhibitory effect of many neurotransmitters (e.g., via M2/M4 muscarinic receptors, alpha(2) adrenoreceptors, micro/delta opioid receptors, GABAB receptors). The G(o) betagamma-subunit acts in two ways: first, and most ubiquitously, by direct binding to CaV2 Ca(2+) channels, resulting in a reduced sensitivity to membrane depolarization and reduced Ca(2+) influx during the terminal action potential; and second, through a direct inhibitory effect on the transmitter release machinery, by binding to proteins of the SNARE complex. G(s) and G(q) are mainly responsible for receptor-mediated facilitatory effects, through activation of target enzymes (adenylate cyclase, AC and phospholipase-C, PLC respectively) by the GTP-bound alpha-subunits.

Lamotrigine inhibition of glutamate release from isolated cerebrocortical nerve terminals (synaptosomes) by suppression of voltage-activated calcium channel activity

Lamotrigine (LAG) is an antiepileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters. The present study was aimed at investigating the effect of LAG on the 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). LAG inhibited the release of glutamate evoked by 4AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C). In addition, LAG did not alter the resting synaptosomal membrane potential or 4AP-evoked depolarization. Furthermore, ionomycin-evoked glutamate release was not affected by LAG. Based on these results, we suggest that presynaptic calcium influx blockade and inhibition of glutamate release may underlie the mechanism of action of LAG. These action may also contribute to their neuroprotective properties in excitotoxic injury.

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons.

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABAAR)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABAAR activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca2+ influx through L-type voltage-gated Ca2+ channels. We further demonstrate that GABAAR activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABAARs. This BDNF/TrkB-dependent increase in surface levels of GABAARs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABAAR surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABAAR activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes).

Ionotropic glutamate receptor agonists, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release. We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.

Attenuation of Phospholipid Signaling Provides a Novel Mechanism for the Action of Valproic Acid

ABSTRACT Valproic acid (VPA) is used to treat epilepsy and bipolar disorder and to prevent migraine. It is also undergoing trials for cancer therapy. However, the biochemical and molecular biological actions of VPA are poorly understood. Using the social amoeba Dictyostelium discoideum, we show that an acute effect of VPA is the inhibition of chemotactic cell movement, a process partially dependent upon phospholipid signaling. Analysis of this process shows that VPA attenuates the signal-induced translocation of PHCrac-green fluorescent protein from cytosol to membrane, suggesting the inhibition of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) production. Direct labeling of lipids in vivo also shows a reduction in PIP and PIP2 phosphorylation following VPA treatment. We further show that VPA acutely reduces endocytosis and exocytosis—processes previously shown to be dependent upon PIP3 production. These results suggest that in Dictyostelium, VPA rapidly attenuates phospholipid signaling to reduce endocytic trafficking. To examine this effect in a mammalian model, we also tested depolarization-dependent neurotransmitter release in rat nerve terminals, and we show that this process is also suppressed upon application of VPA and an inhibitor of phosphatidylinositol 3-kinase. Although a more comprehensive analysis of the effect of VPA on lipid signaling will be necessary in mammalian systems, these results suggest that VPA may function to reduce phospholipid signaling processes and thus may provide a novel therapeutic effect for this drug.

Kainate receptors with a metabotropic modus operandi.

Kainate receptors (KARs), together with AMPA and NMDA, are typically described as ionotropic glutamate receptors. The functions of KARs have begun to be elucidated only in the last decade. Although some the actions of KARs are classically ionotropic, surprisingly others seem to involve the activation of second-messenger cascades and invoke metabotropic roles for this type of glutamate receptor. In this review, we describe these metabotropic actions of KARs in relation to the putative signalling cascades involved. Although it is still a mystery how KARs activate G proteins to stimulate second-messenger cascades, intriguingly, in very recent studies, specific subunits of KARs have been demonstrated to associate with G proteins. Altogether, the body of evidence supports the hypothesis that, together with the canonical ionotropic operation, KARs expedite long-lasting signalling by novel metabotropic modes of action.

Metabotropic actions of kainate receptors in the CNS

Kainate receptors (KARs), together with NMDA and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors (AMPA), are typically described as ionotropic glutamate receptors. Although ionotropic functions for KARs are beginning to be characterized in multiple brain regions, both, in the pre‐ and post‐synaptic compartments of the synapse, there is accumulating evidence that KARs mediate some of their effects without invoking ion‐fluxes. Thus, since 1998, when the first metabotropic action of KARs was described in the modulation of GABA release in hippocampal interneurons, there have been increasing reports that some of the functions of KARs involve the participation of intracellular signalling cascades and depend on G protein activation. These surprising observations, attesting metabotropic actions of KARs, akin to those usually attributed to seven transmembrane region G protein‐coupled receptors, make the physiological classification and description of glutamate receptors more complex. In the present review, we describe the metabotropic roles of KARs in the CNS and discuss the intriguing properties of this receptor which, structurally shows all the facets of a typical ionotropic receptor, but appears to express a metabotropic remit at some key synapses.

Loss of phosphatidylinositol 4-kinase 2α activity causes late onset degeneration of spinal cord axons

Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2α isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2α kinase activity initially appear normal. However, adult Pi4k2aGT/GT animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2aGT/GT animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.